Obesity-induced resistance to insulin action is the primary pathophysiological defect that predisposes to type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). Because current management options remain limited, identification of new regulatory mechanisms that govern the metabolic response to insulin should serve to identify novel opportunities for pharmacologic intervention. The objective of this research is to explore the role of membrane phosphatidylcholine (PC) composition in the regulation of hepatic lipid and glucose metabolism. The rationale is that the identification of a novel mechanism that regulates hepatic insulin sensitivity could lead to the identification of new therapeutic targets for the treatment of NAFLD and type 2 diabetes. Guided by extensive preliminary data, the central hypothesis of this research plan is that phosphatidylcholine transfer protein (PC-TP) functions as a sensor of PC molecular species and controls glucose homeostasis by forming a regulatory complex together with thioesterase superfamily member 2 (THEM2) and tuberous sclerosis complex 2 (TSC2), which modulates insulin signaling potentialy via endoplasmic reticulum (ER) stress. This will be tested in two specific aims: 1) Define the mechanisms by which PC-TP and THEM2 regulate insulin signaling through TSC2 interactions; 2) Determine the role of PC molecular species on binding and activation of THEM2 and TSC2 by PC-TP. In aim 1, the effect of siRNA-mediated silencing of PC-TP and THEM2 expression on insulin signaling will be determined in cell culture systems by measuring the activity of key effectors. The role of TSC2 in the PC-TP- and THEM2-mediated regulation of insulin signaling will be probed in cell lines lacking TSC2 expression. The potential roles of PC-TP and THEM2 in the induction of ER stress will be studied using chemical reagent tunicamycin in cultured cells and prolonged high-fat diet in Pctp-/- and Them2-/- mice. It is anticipated that PC-TP and THEM2 will regulate hepatocellular insulin signaling directly both by forming a novel protein complex with TSC2 and by modulating ER stress. This is important because increased ER stress is associated with development of insulin resistance in association with obesity. In aim2, the influence of PC molecular species on the interactions between purified recombinant PC-TP and THEM2 or TSC2 will be examined in vitro by puldown assays and by surface plasmon resonance. The efect of the PC molecular species bound to PC-TP on the activity regulation of THEM2 and TCS2 will be detected in vitro by measuring their fatty acyl-CoA thioesterase and GAP activity, respectively. We expect that polyunsaturated PC molecular species bound to PC-TP will promote interactions with THEM2 and TSC2 and stimulate the activity of these proteins. Overall, this proposal will elucidate mechanisms by which membrane phospholipid composition regulates glucose metabolism, which is significant because the faty acyl composition of the membrane phosphatidylcholines varies in health and disease. These studies are expected to identify new therapeutic targets for the management of type 2 diabetes and NAFLD.